The In Vivo Architecture of the Exocyst Provides Structural Basis for Exocytosis

Abstract

Graphical AbstractHighlights d An integrative approach reconstructs protein complexes in 3D through live-cell imaging d We use this approach to reconstruct the exocyst complex bound to a vesicle in vivo d Exocyst is a stable complex and regulatory proteins target its multimerization site d We model how exocyst binds the vesicle allowing its contact with the plasma membrane In Brief Microscopy-derived spatial constraints allow modeling of the exocyst structure in vivo. Exocyst Plasma membrane Computer model by trilateration of distances Secretory vesicle 10 nm Distance measurements by fluorescence microscopy PICT Protein complex Anchor site GFP RFP FRB FKBP 1 μm SUMMARYThe structural characterization of protein complexes in their native environment is challenging but crucial for understanding the mechanisms that mediate cellular processes. We developed an integrative approach to reconstruct the 3D architecture of protein complexes in vivo. We applied this approach to the exocyst, a hetero-octameric complex of unknown structure that is thought to tether secretory vesicles during exocytosis with a poorly understood mechanism. We engineered yeast cells to anchor the exocyst on defined landmarks and determined the position of its subunit termini at nanometer precision using fluorescence microscopy. We then integrated these positions with the structural properties of the subunits to reconstruct the exocyst together with a vesicle bound to it. The exocyst has an open hand conformation made of rod-shaped subunits that are interlaced in the core. The exocyst architecture explains how the complex can tether secretory vesicles, placing them in direct contact with the plasma membrane.

The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

“…To investigate the formation and disruption of Pol I transcription complexes in vivo, we used PICT (Protein interactions from Imaging Complexes after Translocation), a fluorescence microscopy technique to analyse protein interactions in living cells (Gallego et al, 2013; Picco et al, 2017). This technique uses cellular anchoring platforms tagged with both RFP and FK506-binding protein (anchor-RFP-FKBP) to recruit proteins tagged with FKBP-binding domain (bait-FRB).…”

Identifying direct contacts between protein complex subunits from their conditional dependence in proteomics datasets

“…[54] and Picco et al . [55] have attempted to resolve the yeast exocyst subunit connectivity map using co-purification and nanometer precision fluorescence microscopy, respectively. Interestingly, Heider and colleagues identified two sub-complexes, sub-complex I consisting of Sec3/EXOC1 (denoting yeast/human orthologs), Sec5/EXOC2, Sec6/EXOC3, Sec8/EXOC4 and sub-complex II consisting of Sec15/EXOC6, Sec10/EXOC5, Exo84/EXOC8 and Exo70/EXOC7.…”

AFM/TIRF force clamp measurements of neurosecretory vesicle tethers reveal characteristic unfolding steps

“…Most recently, a model was proposed for the architecture of the exocyst and it was suggested that vesicles may be tethered by multiple exocyst complexes forming a ring surrounding the vesicle-plasma membrane contact zone where fusion occurs [33]. The multiplicity of tether extension events in the pulling experiments described here, may thus involve unfolding events from multiple exocyst complexes tethering a secretory vesicle.…”